The focus of our research effort will be to identify membrane components involved in mediating the action of insulin on hexose transport subsequent to binding of the hormone to cell surface receptors. Two chemical clues to the identity of such regulatory components of membrane transport which we plan to exploit are: 1) the strong evidence that glucocorticoids inhibit in vitro the synthesis of fat cell membrane protein(s) involved in the regulation of the hexose transport system, and 2) our recent findings which indicate that membrane sulfhydryls play an important role in triggering the activation of the transport system by insulin in fat cells. We hope to identify the membrane proteins or glycoproteins implicated above by specific labelling techniques followed by SDS gel electrophoresis of the plasma membranes. Further, we plan to probe intact fat cells with agents which react with chemically specific protein moieties in the presence or absence of insulin and to analyze the labelling pattern of membrane components resolved on SDS gels. Possible topographical or protein conformational changes elicited in membrane proteins by insulin may be detectable as a difference in the amount of label in membrane peptide(s). In addition, we propose to use a new filtration procedure which directly monitors (H3)-3-O-methylglucose transport in brown fat cells to screen for agents which block insulin action on transport but not control transport rates or insulin binding to cell receptors. The chemical specificity of such agents may be useful in identifying membrane moieties involved in insulin-activated transport. We will also use this new transport assay to define natural regulators of membrane transport by testing the effects of such agents as nucleotides, fatty acids, metabolic intermediates, and other natural products, and studying their possible roles in insulin action.